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. 2015 Nov 2;5:15916. doi: 10.1038/srep15916

Figure 1. Production of farnesylated and methylated KRAS4b.

Figure 1

(a) Pathway of KRAS4b processing in eukaryotic cells. Farnesyl pyrophosphate (FARN-PP) is transferred to Cys185 of KRAS4b by farnesyl transferase (FNT; composed of subunits A and B). After transport to the cytoplasmic face of the ER membrane, the 3 amino acids at the C-terminus of KRAS4b are removed by Ras converting enzyme 1 (RCE1) and the terminal carboxylate is methylated by isoprenylcysteine carboxyl methytransferase (ICMT) in a reaction that uses S-adenosylmethionine (AdoMet) and produces S-adenosylhomocysteine (AdoHcy). After methylation, the fully processed KRAS is trafficked to the cytoplasmic face of the plasma membrane. (b) Purification scheme of processed KRAS4b using Trichoplusia ni (T. ni) insect cells as the expression host. (c) SDS-PAGE analysis of purification. M – molecular weight standards; L – soluble lysate; I – pool from initial IMAC; S – SP sepharose pool; T – Tev protease digestion; F – Final protein from second IMAC. (d) ESI-MS analysis of final protein (e) MALDI-TOF MS/MS analysis of peptides derived from GluC-digested processed KRAS4b confirming the C-terminal peptide is farnesylated and methylated.